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Molecular typing of somatic coliphages to determine their presence
and survival in environmental waters
Hee Suk Lee1, Jeremy Chemla1, Thomas A Randall2 and Mark D. Sobsey1
1: Department of Environmental Sciences and Engineering, University of North Carolina at Chapel
Hill, Chapel Hill, NC, USA
2: Center for Bioinformatics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Keywords: Group-specific PCR, somatic coliphage, sewage, environmental water, persistence
INTRODUCTION
Public health risks posed by enteric viruses in water have led to the search for
reliable indicators of them in drinking and recreational waters. Culture and molecular
methods are available to detect enteric viruses in water, but they are difficult,
expensive and slow to yield results when attempting to detect all enteric viruses.
Coliphages are viruses of E. coli which is a bacterial indicator of fecal contamination
of water. Many waterborne pathogens are enteric viruses, so a bacterial indicator
alone may be inadequate or unreliable as indicator for estimating enteric virus
presence in contaminated water. Somatic coliphages have long been proposed as viral
indicators in sewage and water because they are abundant and often outnumber malespecific coliphages and human enteric viruses (Kott, 1966, Dhillon et al., 1970;
Dhillon and Dhillon, 1972; Moce-Llivina et al., 2005, Jofre, 2008). Somatic
coliphages are heterogeneous, encompassing four families: Myoviridae, Siphoviridae,
Podoviridae, Microviridae, and several genera, with differences in DNA content, size,
structure, and life cycle, ranging from tailed, double-stranded DNA phages of the
order Caudoviriales to the small, cubic Microviridae containing single-stranded DNA
(The Bacteriophage, 2nd ed., Chapter 2, 2006). In order to identify a candidate somatic
coliphage family for use as an indicator for the presence of human enteric viruses in
sewage and water, we developed nucleic acid-based molecular methods to detect
specific somatic coliphage taxonomic groups and used them to evaluate
thecomparative prevalence and persistence of different somatic coliphage families in
sewage.
METHODS
Wild-type somatic coliphage survival was determined in primary sewage effluent
from the Orange County Water and Sewer Authority (OWASA) wastewater treatment
plant, Chapel Hill, NC. Sewage effluent aliquots were held at room (23-25°C) and
refrigerator (4°C) temperatures. Samples were taken periodically and assayed for
virus infectivity by Single Agar Layer (SAL) using E. coli CN-13 host. At each time
point, representative numbers of somatic coliphage plaques (20-50) were chosen
randomly from SAL plates for molecular identification.
Oligonucleotide primers targeting each somatic coliphage family were developed
using bioinformatics tools for sequence analysis conducted on the full genome
sequences of 9 strains of Myoviridae, 44 strains of Microviridae, 8 strains of
Podoviridae and 9 strains of Siphoviridae from the National Center for Biotechnology
Information website. Sequence analysis was done using Vector NTI (ver.10,
Invitrogen), MEGA (version 4, Tamura et al., 2007), and Jalview (version 2,
University of Dundee). Multiple alignments were carried out with ClustalW2, and
blast searches of protein-protein in the Myoviridae and Siphoviridae families were
done to find conserved protein regions among strains for family-specific primers.
PCR primers and amplification conditions were developed and verified for familyspecific PCR of each somatic coliphage family (data not shown).
RESULTS AND CONCLUSIONS
Experiments in which test waters (reagent grade water and surface water) were
spiked with prototype positive strains and incubated at 4 and 25°C showed that
Myoviridae as represented by T4, Microviridae as represented by PhiX174, and
Siphoviridae as represented by Lambda survived the longest at both temperatures.
[Data are not shown in this article] From a time-course study of somatic coliphage
survival in primary sewage effluent incubated at 25°C and 4°C, a total of 275
presumptive somatic coliphage isolates were collected and archived for subsequent
taxonomic characterization. By applying group-specific conventional PCR for each of
the 4 major taxonomic families, it was possible to document the composition of the
somatic coliphage population in primary sewage effluent and how it changed over
time. The majority of identifiable somatic coliphages isolates from primary sewage
effluent belonged to the Microviridae family, some isolates belonged to the
Myoviridae family, and no isolates were detected from the Siphoviridae or the
Podoviridae families. The Microviridae family became even more prevalent in the
sewage population after several weeks of incubation at 4 and 25°C (Table 1.1).
Table 1.1 Somatic coliphage families of primary sewage effluent isolates by incubation temperature
and holding time using family-specific primers for PCR analysis
Days (Temperature)
0-1
29(25°C)
47(25°C)
65(4°C)
65(25°C)
Microviridae (Pos./Total)
10/48
22/26
31/36
31/47
3/3
Myoviridae (Pos./Total)
2/48
1/26
0/36
0/47
0/3
Siphoviridae (Pos./Total)
0/48
0/26
0/36
0/47
0/3
Podoviridae (Pos./Total)
0/48
0/26
0/36
0/47
0/3
By conventional, group-specific PCR methods developed to identify each of the 4
major taxonomic groups of somatic coliphages (Microviridae, Myoviridae,
Siphoviridae, and Podoviridae), it was possible to detect and classify individual
coliphage isolates in sewage. Based on prevalence and persistence, the Microviridae
family appears to be a promising candidate somatic coliphage indicator group of
sewage contamination and the possible presence of human enteric viruses in fecally
contaminated water. The development of PCR-based molecular typing of somatic
coliphage families provides new and improved tools to study their ecology and
environmental distribution, presence in sewage and water and response to treatment
processes as viral indicators of drinking water and its sources and recreational water.
References
Dhillon TS, Chan YS, Sun SM, Chau WS 1970 Distribution of coliphages in Hong Kong sewage Appl Microbiol
Aug;20(2): 187-91
Dhillon T.S, Dhillon E.K. 1972 Studies on bacteriophage distribution. II. Isolation and host rage based
classification of phages active on three species of Enterobacteriaceae. Jpn J Microbila. Jul;16(4):297-306
Jofre, J. 2008 Is the replication of somatic coliphages in water environmental significant? J.Appl. Microbial.
106:1059-1069
Kott, Y. Rose, N., Sperber S., and Betzer, N. 1974 Bacteriophages as viral pollution indicators Water Research
8:165-171
Moce-Llivina, L., Lucena, F., and Jofre, J. 2005 Enteroviruses and bacteriophages in bathing waters Appl. Environ
Microbiol 71(11): 6838-6844
Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA)
software version 4.0 Molecular Biology and Evolution 24:1596-1599.
Response to Reviewers' comments:
Reviewer #3: The following items are not clear in the abstract, so information and
logical explanation should be arranged again for the presentation.
# (1) The relationship between coliphages and enteric viruses,
(Add in article) (Response #1) Coliphages are viruses of E. coli which is a bacterial
indicator of fecal contamination of water. Many waterborne pathogens are enteric
viruses, so a bacterial indicator alone may be inadequate or unreliable as indicator for
estimating enteric virus presence in contaminated water.
Coliphages are viruses of E. coli which is a bacterial indicator of fecal contamination of water.
Many waterborne pathogens are enteric viruses, so a bacterial indicator alone may be
inadequate or unreliable an an indicator for estimating enteric virus presence in
contaminated water. Stetler et al. (1984), found that coliphages showed better
correlation with enteric virus presence than total coliforms, fecal coliforms, fecal
streptococci, or standard plate count organisms. Male-specific coliphages have been
extensively studied by a number of researchers as viral indicators, based on their
morphological similarity to enteric viruses. However, some somatic coliphages also may
be useful and effective as viral indicators of enteric viruses.
# (2) The stability of each family in sewage and water,
(Add in article) (Response #2) Experiments in which test waters (reagent grade water
and surface water) were spiked with prototype positive strains and incubated at 4 and 25°C
showed that Myoviridae as represented by T4, Microviridae as represented by PhiX174, and
Siphoviridae as represented by Lambda survived the longest at both temperatures. [Data are
not shown in this article]
To investigate the possibility of individual somatic coliphage families (Microviridae,
Myoviridae, Siphoviridae, and Podoviridae) serving as reliable viral indicators in
environmental waters, survival tests were performed using prototype strains to determine
if the families differ in their relative persistence in water. Experiments in which test
waters (reagent grade water and surface water) were spiked with prototype positive
strains and incubated at 4 and 25°C showed that Myoviridae as represented by T4,
Microviridae as represented by PhiX174, and Siphoviridae as represented by Lambda
survived the longest at both temperatures. [Data are not shown in this article] However,
there are no stability data of each family in sewage water.
# (3) The accommodation of the growth ability in this culture condition,
(Explain here and no extra adding in article)
Table shows the concentrations of total somatic coliphages (all genogroups) in sewage
samples over time at 4°C and 25 °C. The concentrations of total somatic coliphages
decreased over time at both temperatures. However, the decline over time at 4°C was
less than at 25°C. At both temperatures, some phages underwent a decline after day 28.
After day 65, the concentration total of somatic coliphages at 25°C had declined to 0.1
PFU/mL, so only three somatic coliphage isolates were collected at this time period.
[Data are not shown in this article]
Table Survival of somatic coliphages in primary effluent sewage over time, and number of somatic
coliphage isolates as picked plaques enriched at each sample time point at temperatures of 4 °C and 25
°C)
Change of titer
(Unit: PFU2/ mL)
Temperature
(°C)
4
Number of Isolates
1:
N/A: data not available
2:
day0
day10
day15
day29
day47
day65
400
170
141
23
N/A
32
25
400
N/A
67
6
10
0.1
4
25
48
30
N/A
29
30
26
26
N/A
36
47
3
PFU: Plaque Forming Units
# (4) The reason for identifying Microviridae family as enteric virus indicator.
(Explain here and no extra adding in article)
The results of studies on prototype somatic coliphage survival in seeded waters suggest that
based on their greater persistence in water over time, T4 in the Myoviridae, PhiX174 in the
Microviridae, and Lambda in the Siphoviridae are possible candidates as fecal indicator
viruses in water due to their greater survival compared to T1 in the Siphoviridae and T7 in the
Podoviridae. [Data are not shown in this article]
The Microviridae family became even more prevalent in the sewage population after several
weeks of incubation at 4 and 25°C in this study. So, based on prevalence and persistence,
the Microviridae family appears to be a promising candidate somatic coliphage indicator
virus group of sewage contamination and the possible presence of human enteric viruses
in fecally contaminated water.
This sentence is already mentioned in this article.